GSM8084233: MNase-TGFb; Mus musculus; MNase-Seq
Samples containing 1x107 cells were were washed twice with cold PBS and cell pellets were resuspended with cold MNase-RSB (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM MgCl2). After incubation on ice for 10 min, 10% Nonidet P-40 was added to make a final 0.5% Nonidet P-40 and cells were homogenously mixed by inverting. Nuclei were pelleted at 1300xg for 5 min and resuspended with MNase-digestion buffer (10 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2, 250 mM sucrose, 0.5 mM dithiothreitol, DTT). Nuclei were incubated with varying concentrations of MNase to optimize the fragmentation (~60% mononucleosome, ~20% dinucleosome, and ~10% trinucleosome). We treated nuclei with 100 U of MNase (Thermo Fisher Scientific, Cat #: 88216). After incubation for 5 min at 37ºC with mixing, stop solution (1% SDS, 100 mM NaHCO3, 20 mM EDTA) was added and incubated at 65ºC for 90 min. DNA fragments were incubated with Tris-HCl pH 7.5, Proteinase K and Glycoblue (Thermo Fisher Scientific, Cat #: AM9515) for 16 h at 65ºC. Mononucleosomal DNA (~147 bp) was extracted using QIAquick Gel Extraction Kit (Qiagen). Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit according to the manufacturer's instructions with 0.02-5 ng input DNA and 8-14 cycles of PCR.